Dihydroxythiophenes Are Novel Potent Inhibitors of Human Immunodeficiency Virus Integrase with a Diketo Acid-Like Pharmacophore

Kehlenbeck, S.; Betz, Ulrich; Birkmann, Alexander; Fast, B.; Goller, Achim; Henninger, Kerstin; Lowinger, Timothy B.; Marrero, Diana; Paessens, A; Paulsen, D.; Pevzner, V.; Schohe-Loop, R.; Tsujishita, Hideki; Welker, Reinhold; Kreuter, Jörg; Rübsamen‐Waigmann, Helga; Dittmer, F.

doi:10.1128/jvi.00306-06

Cited by 15 publications

(12 citation statements)

References 53 publications

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“…The N155H, Q148R, and E92Q enzymes were all between 4 and 15% as efficient as WT, with N155H being the weakest single variant (4%). The N155S virus has been reported to be less fit than WT (42,43), and the N155E, N155K, and N155L variants were reported to have 6% of WT strand transfer activity (44), similar to the N155H results reported here. T66I had a relative efficiency of 36%, indicating that this enzyme is not much altered catalytically, as compared with WT, similar to what has been reported previously (38).…”

Section: Discussionsupporting

confidence: 78%

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

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In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (k p /K m ) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (k p ) values. However, smaller secondorder effects were caused by up to 4-fold increases in K m values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

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Section: Discussionsupporting

confidence: 78%

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

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“…Also, Lee and Robinson (55) reported that both G149S and Q148A were 11-and 22-fold resistant, respectively, to the strand transfer inhibitor L-731,988, although certain recently described dihydroxythiophene strand transfer inhibitors appear to bind differently and are not resistant to Q148K (63). A direct interaction between Gln 148 and strand transfer inhibitors has been suggested by several other studies.…”

Section: Discussionmentioning

confidence: 92%

Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

Dicker¹,

Samanta²,

Li³

et al. 2007

Journal of Biological Chemistry

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Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G 2 T 1 C ؊1 A ؊2 ) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G 2 :C 2 base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln 148 interacts with G 2 , T 1 , and C ؊1 at the 5 end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.Integration, the insertion of a double-stranded DNA copy of the viral RNA genome into the host genome, is absolutely required for HIV 2 replication (1). As such, integration presents an attractive target for the development of small molecule inhibitors that could be used to treat HIV. Several integrase (IN) inhibitors that are progressing through clinical development (2, 3) have been shown to lower viral load in infected patients.The virus-encoded IN protein catalyzes two essential activities in the viral life cycle, 3Ј-processing and strand transfer. The 3Ј-processing reaction cleaves off the final two bases (5Ј-GT dinucleotide) from the 3Ј ends of the viral LTR. The strand transfer activity catalyzes the concerted insertion of the two viral 3Ј ends into the host genome with a 5-bp separation. To accomplish this, IN simultaneously positions the two 3Ј-hydroxyls of the LTRs for nucleophilic attack onto the phosphodiester bonds of the genomic DNA (4). In vitro, both reactions are catalyzed by divalent magnesium or manganese, although magnesium is thought to be the actual metal cofactor in cells (5).Suitable in vitro systems for studying these processes have been described, in which the full-length protein is minimally required to direct both 3Ј-processing and strand transfer (6, 7). In these systems, IN is typically allowed to form an in situ complex with a model D...

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“…Notably the recombinant virus containing V151I, contrary to a recent study (37), did not demonstrate any decrease in susceptibility to any compound. The effect of V151I on susceptibility is inconsistent and may depend on the plasmid backbone used (15,19,25). It is also possible that other integrase polymorphisms within the isolate might have altered its response to the InSTI tested.…”

Section: Vol 53 2009 Hiv Integrase and Inhibitor Susceptibility Polmentioning

confidence: 99%

Natural Polymorphisms of Human Immunodeficiency Virus Type 1 Integrase and Inherent Susceptibilities to a Panel of Integrase Inhibitors

Low

Prada

Topper

et al. 2009

Antimicrob Agents Chemother

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We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drugresistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance. A phenotypic analysis was done on 10 recombinant viruses derived from nine patients against a panel of six diverse InSTI. Few of the polymorphisms associated with in vitro InSTI resistance were identified in the samples from newly infected individuals or those patients with MDR HIV-1. We identified polymorphisms V72I, L74I, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N. V72I was the most common, seen in 63 (56.3%) of the AHI samples. E157Q was the only naturally occurring mutation thought to contribute to resistance to elvitegravir, raltegravir, and L-870,810. None of the patient-derived viruses demonstrated any significant decrease in susceptibility to the drugs tested. In summary, the integrase coding region contains as much natural variation as that seen in protease, but mutations associated with high-level resistance to existing InSTI are rarely, if ever, present in integrase naïve patients, especially those being used clinically. Most of the highly prevalent polymorphisms have little effect on InSTI susceptibility in the absence of specific primary mutations. Baseline testing for integrase susceptibility in InSTI-naïve patients is not currently warranted.

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Dihydroxythiophenes Are Novel Potent Inhibitors of Human Immunodeficiency Virus Integrase with a Diketo Acid-Like Pharmacophore

Cited by 15 publications

References 53 publications

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

Natural Polymorphisms of Human Immunodeficiency Virus Type 1 Integrase and Inherent Susceptibilities to a Panel of Integrase Inhibitors

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